emerging optical microscopy techniques for electrochemistry
TRANSCRIPT
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Emerging Optical Microscopy Techniques for Electrochemistry
Jean-François Lemineur,a Hui Wang,b Wei Wang,b,* Frédéric Kanoufia,*
a Université de Paris, ITODYS, CNRS, 75006 Paris, France.
b State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry
and Chemical Engineering, Nanjing University, Nanjing 210023, China.
* corresponding authors, e-mails: [email protected], [email protected]
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ABSTRACT
An optical microscope is probably the most intuitive, simple and commonly used
instrument to observe objects and discuss behaviors through images. Although the
idea of imaging electrochemical processes operando by optical microscopy was
initiated 40 years ago, it was not until significant progress made in the last two
decades in advanced optical microscopy or plasmonics that it could become a
mainstream electroanalytical strategy. This review illustrates the potential of different
optical microscopies to visualize and quantify local electrochemical processes with
unprecedented temporal and spatial resolution (below the diffraction limit), up to the
single object level with subnanoparticle or single molecule sensitivity. Developed
through optically and electrochemically active model systems, optical microscopy is
now shifting to materials and configurations focused on real-world electrochemical
applications.
KEYWORDS
Optical microscopy, electroanalysis, electrochemical conversion, single entity
electrochemistry, nanoparticles, operando imaging, spatiotemporal resolution
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1. INTRODUCTION
Electrochemistry is a vivid branch of science, particularly within the search for
renewable energy sources and systems enabling the conversion and storage of energy.
While great efforts have been made toward the synthesis and processing of
electroactive and electrocatalytic materials, often emphasizing the importance of their
structuring at the nanoscale, the improvement of the performance of most
electrochemical devices is hampered by the kinetic limitations of electrochemical
reactions. The understanding of their mechanisms and fundamentals relies on the
establishment of structure-function relationships, particularly at the nanoscale. This
has then driven the shift of traditional electroanalytical strategies and techniques
based on ensemble-averaged methods, e.g., current-potential, response toward the
imaging of electrochemical processes with higher sensitivity, spatial and temporal
resolution and manyfold complementary information.
Despite considerable progress in advanced in situ/operando characterization
techniques, optical microscopy remains the only technique that requires simple
operating procedures while being noninvasive and enabling multiple instrumental
couplings. Optical imaging of electrochemical processes was introduced in the
mid-1980s (1) along with scanning electrochemical microscopy. It was not until the
improvement of optical detectors (and components) and the development of
plasmonics that electroanalytical strategies employing optical microscopy were
brought back to the fore. The recent concepts of superlocalization, allowing to track
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phenomena with a resolution of a few nanometers which is lower than the smallest
picture element, i.e., the pixel, open many perspectives for imaging electrochemical
processes beyond the diffraction limit. Several reviews have detailed the general
operating principles and applications of such advanced optical microscopies in
(nano)chemistry, sometimes in electrochemistry (2–5). Herein, we summarize their
recent achievements in the imaging of multifarious electrochemical systems. After a
short description of some of the microscopes used, we show how they are currently
employed to resolve and quantify the heterogeneity of electrochemical interfaces,
from the macroscopic scale to the single nanoobject or even to the subentity or single
molecule level.
2. OPTICAL MICROSCOPES
A detailed description of the operating principle and configurations of the various
optical microscopes employed in electrochemistry can be found in (2–5). This review
mostly focuses on the use of wide-field microscopes, in which the light emanating
from a whole substrate is collected by a microscope objective and captured in a single
snapshot by a camera or a spectrograph for spectroscopic imaging. They offer higher
spatiotemporal resolution imaging than point scanning, e.g., confocal or tip-enhanced
Raman scattering, microscopes: within a single >50x50µm2 image snapshot, within
millisecond timescale, thousands of localized electrochemical behaviors can be
simultaneously obtained.
These microscopes are sensitive to the optical properties of the sample of interest,
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mostly absorbance, refractive index, scattering or luminescence. The growing
popularity of optical microscopy approaches in electrochemistry is not only related to
the ability to image but also the collection of quantitative information from the
mathematical treatment of the optical signal; see, e.g., (3). Plasmonic metals, e.g., Au
or Ag, constitute a highly sensitive detection tool in optics, as their interaction with
light induces the surface-confined oscillation of their free electrons known as surface
plasmon resonance (SPR). The SPR is strongly sensitive to the metal local charge
density or the refractive index of its environment, enabling different plasmonic-based
imaging of electrochemical processes. In SPR microscopy, the light locally reflected
by the interface between a thin layer of Au (used as an electrode) and an electrolyte
produces an SPR image sensitive to a wide variety of (electro)chemical reactions (2,
6). Localized SPRs, or LSPRs, are supported by plasmonic nanoparticles, NPs, or
nanostructures. Tracking the scattering (or LSPR) spectra of single plasmonic NPs
allows sensing single NP electrochemistry (2, 4, 7). The illumination of plasmonic
NPs or nanostructures (roughened electrodes) also produces a strong electromagnetic
near field able to enhance the Raman scattering generated by (individual) molecules
by several orders of magnitude (8, 9). The local increase in Raman intensity is used to
provide molecular vibrational images in surface-enhanced Raman scattering and
SERS microscopy with single molecule sensitivity.
Bright field and reflectivity microscopes mostly use (axial) illumination along the
objective axis and collect light transmitted or reflected by the sample of interest. They
can probe absorbance, refractive index or light emission, such as fluorescence or
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Raman scattering, after appropriate filtering of the excitation light beam. Oblique
incidence illuminations are used mostly for (i) dark-field configurations and (ii)
extreme total internal reflection (TIR) conditions, as they offer a lower optical
background level, enabling single nanoparticle or single molecule imaging sensitivity.
Dark-field illumination avoids blurring the detector, which only collects the light
scattered by the sample of interest. It has mostly been used to image the scattering of
plasmonic NPs with the eventual spectroscopic capture of their LSPR spectrum.
However, a broader class of scattering NPs has more recently been imaged at higher
sensitivity by interferometric scattering microscopes (10).
The TIR condition allows confined illumination (by evanescent waves) to light up
only objects located within a few hundred nm above the illuminated interface, which
is particularly useful for single-molecule fluorescence detection. Similar TIR
illumination conditions are used in plasmonic-based (SPR) microscopy.
Imaging without optical illumination, and therefore at the lowest optical background,
is possible using the electrochemical triggering of a luminescent reaction, named
electrochemiluminescence microscopy (5, 11, 12). As it involves chemically unstable
precursors of the luminescence reaction, electrochemiluminescence offers chemically
confined illumination of objects near the electrode and single photon sensitivity (13).
Finally, a microscope is characterized by two crucial notions: its sensitivity, i.e., its
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ability to detect an object, and its resolution, i.e., its ability to distinguish two objects
close together. Microscopes are diffraction limited, meaning that objects should be
separated by a distance greater than λ/2NA, with λ being the illumination wavelength
and NA being the objective numerical aperture. Furthermore, single objects smaller
than this limit, e.g., single NPs or single molecules, appear in an optical image as an
identical optical pattern, regardless of their structure or composition, named the point
spread function (PSF) or Airy disk. Note that the resolution of localization of optical
microscopes can be greatly improved by image posttreatment consisting of
approximating the PSF by a two-dimensional Gaussian distribution and
algorithmically extracting the spatial origin of a single emitter, also named its optical
center of mass or centroid. By superlocalization approaches, the location of various
electrochemical reactions is visualized operando at single nanoentities with a
resolution <5 nm.
3. OBJECTS OF STUDY
3.1 IMAGING OF HETEROGENEOUS INTERFACES
Probing nanoscale electrochemical events at heterogeneous interfaces discloses the
internal mechanism and detailed dynamics of electron transfer processes in analytical
chemistry and biosensing. Different imaging strategies have been developed to
visualize local electrochemical information at electrode interfaces, such as plasmonic,
electrochemiluminescence, and fluorescence microscopy (1–8). These revolutionary
studies reveal the intrinsic characteristics and mechanisms of nonfaradaic and redox
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processes with ultrasensitive temporal and spatial resolution. This section details the
optical electrochemical imaging of various heterogeneous interfaces.
3.1.1 Individual cells
Monitoring single-cell responses to substrates or small molecules and cellular
processes at the microscopic level deepens the understanding of the mechanisms of
physiological and biochemical dynamics. Optical techniques have been introduced to
study multifarious single-cell electron transfer events with high spatial resolution,
providing detailed information on their transient activities and local distributions (19,
20). Tao et al. first developed plasmonic-based electrochemical impedance
microscopy to uncover heterogeneous processes such as single-cell apoptosis and
electroporation with millisecond time resolution (21). A local impedance
measurement (𝑍) is derived from the local change in plasmonic intensity (∆𝜃) of a
thin Au SPR surface according to 𝑍!!(𝑥,𝑦,𝜔) = 𝑗𝜔𝛼∆𝜃(𝑥,𝑦,𝜔)/∆𝑉, where 𝜔 is
the angular frequency of the AC modulation, x and y are the locations on the electrode,
and 𝛼 is a constant determined by theoretical calculation or experimental calibration.
They optically resolved the local impedance of subcellular structures and ion
distributions in mammalian cells with submicrometer spatial resolution and excellent
detection sensitivity (~2 pS). By combining the electrochemical plasmonic impedance
imaging method with the traditional patch clamp technique (Figure 1a), the fast
propagation of the action potential in individual mammalian neurons was mapped
without any labeling (22). They further investigated the heterogeneous distribution of
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ion channels at the subcellular level and proposed studying various cellular
electrochemical activities and understanding the related biological functions and
mechanisms.
INSERT FIGURE 1
Recently, the groups of Sojic and Paolucci developed a surface-confined microscope
based on electrochemiluminescence illumination of objects and illustrated it to map
membrane adhesion sites of single cells on an electrode (20, 23). Their groups further
demonstrated the influence of photobleaching on electrochemiluminescence emission.
As both photo- and electrochemical activation involve the same excited state, the
more photoactivated the fluorophore is, the less active it is in the
electrochemiluminescence. Despite this issue, new imaging strategies combining
fluorescence recovery and electrochemiluminescence were envisioned (24).
3.1.2 Fingerprints
Visualizing latent fingerprints (LFPs) is an essential method for biometric identity
authentication. Various chemical and physical strategies have been explored to reveal
LFPs, including multimetal immunodeposition, fluorescence, and ink staining (25–30).
The ability of electrochemical techniques to identify explosive residues and other
chemicals secreted by LFPs has gradually gained attention. Tao et al. demonstrated a
plasmonic imaging technique combined with electrochemical measurement to map
human LFPs on an electrode surface (28). Finger secretions block the electron transfer
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process on the electrode, and the plasmonic contrast of the local fingerprint region is
transposed into a local electrochemical current of redox-active molecules in solution.
Later, Su and coauthors (Figure 1b) developed an electrochemiluminescence-based
imaging technique to enhance and visualize local LFPs using different dyes:
Ru(bpy)32+, rubrene, and electropolymerized luminol (25, 27, 29). The
electrochemiluminescence signal was generated only between the ridges of the LFPs,
and different details of the LFPs were resolved: the bifurcation, core, island, pore,
lake, peak, and termini.
Recently, Hu et al. reported a new strategy for transferring and imaging LFPs onto
nonporous substrates using simultaneous interfacial separation of a polydopamine
film and electroless silver deposition (27). As sweat components and underlying
substrates were well preserved, they generalized the approach to different substrates,
regardless of surface hydrophobicity or micromorphology.
3.1.3 Bipolar electrochemistry
A bipolar electrode (BPE) is a conductive material exposed to an external electric
field from the application of a potential difference between two electrodes in an
electrolyte. The potential difference induces electrical polarization at opposite poles
of the bipolar electrode, which manifests as a gradient in the distribution of free
electron density (31). When the potential difference is large enough, opposite
electrochemical reactions occur simultaneously at both ends of the BPE. As these
reactions occur without external current flow, their demonstration requires
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complementary visualizations, such as probe-labeled imaging with
electrochemiluminescence reagents, pH chromogens, or fluorescent dyes, and
label-free plasmonic imaging techniques (32–34). Crooks and coauthors developed
electrochemiluminescence-based imaging of BPE reactions. They provide a means to
locally quantify the thermodynamics and kinetics of the reactions to spatially
reconstruct the voltammogram of these reactions from an electrochemiluminescence
image. A triangular BPE is used, which allows, for electroanalysis, the quantification
of the reaction of interest on the smallest part of the BPE (toward the point of the
triangle) compared to the larger counterelectrode reaction part (35).
Xu and Chen reported an ultrasensitive wireless electrochemiluminescence biosensor
for quantitative monitoring of c-Myc target mRNA in tumor cells on a BPE substrate
(36). In this system-on-chip, they integrated RuSi@Ru(bpy)32+ for optical signal
amplification with a 24-fold improvement over Ru(bpy)32+-NHS labels. Beyond
electrochemiluminescence, Kuhn et al. successively presented other indirect imageries
of BPEs based on pH-triggered local precipitation (37) or fluorescence modulation
(32). The products of these reactions are monitored in the vicinity of the BPE.
Except for the above techniques using optical probes, plasmonic-based microscopy
provides label-free visualization and thus there is no need to engage a faradaic
reaction at the BPE a priori. Wang and coauthors first demonstrated the capability of
plasmonic imaging to directly visualize the interfacial potential distribution on a
bipolar microelectrode array with a sensitivity of 10 mV (33). The external electric
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field controls the redistribution of the free electron density on the BPE and thus
modifies its local dielectric (optical) properties. The local plasmonic response is thus
predicted using the Drude model. Furthermore, it is possible to locate the
zero-potential line on BPEs, where no reaction occurs, regardless of their geometry
(e.g., round, triangular, hexagonal, star, and diamond shapes) during nonfaradaic
charging and faradaic processes (38). The results revealed that the geometry of the
electrode and the nature and redox potential of the faradaic reactions affect the
position of the zero-potential line on the BPE.
3.1.4 Two-dimensional nanomaterials
Two-dimensional (2D) nanomaterials are emerging as novel platforms for
optoelectronics and biosensing due to their unique physical, chemical, and electronic
characteristics (39–41). The spatial charge distribution of these thin layers has been
studied by optical techniques, such as plasmonic, bright field, or interference
scattering microscopy, coupled with electrochemical measurements (6, 41–43). The
optical readout reveals fundamental electrochemical parameters of 2D electrodes and
their heterogeneity, such as quantum capacitance and local charge density, with high
spatial and temporal resolution. Graphene is the most studied 2D material without a
bandgap. Tao and coauthors mapped local electron and hole puddles with charged
impurities in a graphene monolayer by plasmon-based impedance microscopy (42).
The surface charge density, ∆𝜎, is related to the local interfacial capacity per unit
area ( 𝑐 ) and the applied potential (∆𝑉 ), according to ∆𝜎 = 𝑐 ∙ ∆𝑉 . Periodic
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modulations of the potential control the surface charge, resulting in a modulation of
the plasmonic intensity (∆𝜃 ). From the latter ∆𝜃 , extracted from the Fourier
transform of the graphene region images, a local capacitance distribution is obtained
according to 𝑐~∆𝜃/∆𝑉. Further charging induces an expansion of the graphene
according to Pauli repulsion. This expansion is imaged using a nm-sensitive optical
edge-tracking method (44). The technique allows determining the electromechanical
stress that increases quadratically with the modulation of the applied potential and
extracting the Young's modulus of different regions. Further oxidation of graphene at
potentials > 1.4 V results in its conversion to graphene oxide. This process was
imaged in situ by a label-free refractive index-sensitive optical technique such as
interference reflection microscopy (IRM). This reveals the formation of flower-like
patterns from which the local degree of graphene oxidation can be quantified and its
chemical vs. electrochemical oxidation compared (43).
Apart from graphene, molybdenum disulfide (MoS2) monolayers are another
attractive 2D material for next-generation nanoelectronic devices, with a direct
bandgap of 1.9 eV. Tao et al. imaged the local charge distribution of atomically thin
MoS2 upon electrochemical charging. The change in charge induces a local change in
the absorption of MoS2, which is imaged by bright-field transmission microscopy
with higher sensitivity (45).
3.1.5 Electrocorrosion and electrodeposition at a large interface
Electrocorrosion and electrodeposition are classical strategies for fabricating
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functional interfaces and improving the surface characteristics of metallic, ceramic, or
polymeric materials (46, 47). The basic principle of electrocorrosion and
electrodeposition is the destruction and formation of materials on a working electrode
immersed in an electrolyte solution and subjected to an external potential. The
morphological evolution of the electrode surface during these interfacial engineering
processes is essential to uncover the detailed dynamics and accurately determine
structure-function relationships. Different optical imaging techniques have been used
to probe corrosion processes, e.g., fluorescence, reflectivity or confocal microscopy.
They allow the identification of locally different reaction products or solution pH or
the identification and measurement of the size of crevices. V. Pérez-Herranz
developed a wide field reflectivity microscope allowing real-time observation at the
scale of several cm2 on copper and stainless steel electrode surfaces (48). They also
mapped the different corrosion behaviors of crevices and grain boundaries and
identified the generation of gas bubbles. Smyrl et al. used fluorescence microscopy to
image the regions where oxides, which trap fluorophores, preferentially form at
higher resolution. The measurement is complemented by confocal measurements
allowing a topographic (3D) image of crevices (49, 50). Vivier and coauthors
proposed a quantitative assessment by reflectivity imaging of the thickness of passive
layers during corrosion of carbon steel under polarization (51). They performed local
reflectivity measurements of the steel surface during cathodic and anodic
polarizations to study the formation of Fe2O3 and Fe3O4 oxides. It allows the
identification of the most active regions of the surface and the establishment,
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simultaneously over each µm2 regions of the mm2 imaged surface, of local
voltammograms of their activity or transformation.
These same imaging techniques are also used to follow operando the
electrodeposition processes at micrometric scales, in particular in energy storage or
conversion systems (52), to identify the formation of dendrites (53, 54) localized
operando at nanometer resolution (Figure 1c) or electrode passivation (55) and to
remedy them.
3.2 SINGLE NANO-ENTITY STUDIES
The growing use of nanoscale objects is bound to the identification of their intrinsic
properties, for which quantitative nanostructure-activity relationships are urgently
needed. In electrochemistry, different cross-reading approaches have been proposed at
the single NP level (56–59), mainly based on their isolation in time, one
electrochemical event at a time, or in space, by local electrochemical probing. In
addition to probing local electrochemistry with nanoelectrodes or nanopipettes, in the
so-called scanning electrochemical (SECM), electrochemical cell (SECCM) or ion
conductance (SICM) microscopies configurations, one can use optical microscopies
that allow high-sensitivity imaging and detection at the resolution of a single NP.
Recent developments proposed to integrate optical microscopy readout with such
scanning electrochemical microscopes (60, 61). The coupling of optical microscopy
with SECCM is of particular interest to image the subtle electrochemical processes
occurring inside the nano- or microelectrochemical cell, obtained by the confinement
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of a droplet of electrolyte by a nano- or micropipette. Indeed, in addition to high
resolution electrochemical imaging, SECCM allows a high throughput exploration of
local electrochemical processes by a versatile modifications or benchmarking in each
droplet of experimental parameters, e.g. surface or solution composition or
electrochemical interrogation. While SECCM enables nanoscale electrochemical
exploration with single droplet resolution, optical microscopy affords a
complementary subdroplet imaging resolution.
Coupling electrochemistry to optical microscopies appears relevant to probe operando
nanoscale electroactivities. Optical movies allow high-throughput readout of
individual NPs within large ensembles, altogether submitted to the same experimental
condition, allowing identification of subpopulation behaviors, for example by drawing
and comparing their individual electrochemical activity (e.g. current-potential,
charge-time, etc. curves). Moreover, NPs can be differentiated by their size, structure
or composition from their different optical properties, typically their optical cross
section (related to their refractive index, absorption, luminescence, etc.).
In the field of NP electrochemistry, optical microscopy has been applied to reveal and
study a wide variety of chemical or physical processes illustrated in Figure 2a for the
particular case of Ag-based NPs, i.e., the most studied system due to their plasmonic
activity and easily tunable (electro)chemical activity.
INSERT FIGURE 2
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3.2.1 Electrodeposition and electrodissolution
Optical monitoring of an electrode surface during electrodeposition reactions allows
simultaneous capture of the moment and location of formation of individual NPs with
a very large field of view (up to millions of NPs simultaneously (62)) and camera
temporal resolution (up to >1 kHz). Optical imaging then provides statistically
significant data to test and enrich NP nucleation/growth mechanisms and models.
For noble metals, e.g., Ag, the differences in the onset of NP appearance on the
electrode reflect the variability of their nucleation barrier (63, 64). Iron group metals
also reveal competition with other electrode reactions, such as water reduction in the
case of Ni or Co (65, 66). Beyond the local chemical information, localization of the
nucleation sites (67, 68) allowed reconstruction of each diffusion zone around the NPs
and probing diffusion cross-talk between neighboring sites.
Within a region of interest (ROI) centered on each NP, the transient evolution of the
local optical intensity is extracted from optical movies. Such transient gives insights
into the modes and kinetics of single-NP growth (69). This can be converted into the
amount of locally electrodeposited material (63, 64, 70) due to a calibration between
NP size and optical intensity obtained from the optical images of gauge NPs, ex situ
correlative SEM analysis or optical modeling. Combined with Faraday's law, local
currents, in the form of optovoltammograms (Figure 2b), associated with the
growth/dissolution of each NP are obtained by this quantitative analysis, again
evaluated for hundreds of NPs simultaneously.
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The reverse electrodissolution reaction was also studied for electrodeposited NPs (63,
64, 71) or nanocolloids immobilized on an electrode (69, 72, 73). The disappearance
of the optical feature associated with the NP in the images accounts for its dissolution
dynamics, investigated for metallic Ag NPs in different electrolytes (63, 64, 71).
Electrodeposition/stripping is an attractive strategy to decorate electrodes with a high
density of NPs of controlled size distribution, allowing the examination of
structure-activity relationships. The effect of NP size on their oxidation potentials,
observed for Ag NPs, validates Plieth's theory that links the electrochemical stability
of NPs (below ~50 nm) to their surface tension (63, 64).
The electrodissolution of Brownian nanocolloids was also probed by optical
microscopy. It allows tracking the motion of Brownian NPs in solution during their
collision (reactive or not) with a polarized electrode and complements electrochemical
nanoimpact experiments (56), in which the current spikes associated with a reactive
collision of NPs provide information on their size, dispersion, stability, concentration,
etc. The correlated optical and electrochemical detections revealed a more complex
picture. 3D optical tracking of Ag NPs near a polarized interface revealed intermittent
NP-electrode interactions associated with partial oxidation events (72, 74). This
supports the hypothesis of multistep Ag→Ag+ electrodissolution, first established
from high-frequency current traces and attributed to stochastic electrical
disconnection (75). Under conditions favoring Ag+ precipitation, a time lag is
observed between the injection of electrochemical charges and the dissolution of
optically probed NPs, highlighting solid phase conversion (e.g., to oxide, halide, or
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thiocyanate crystals (72, 73, 76–78)).
This type of optical imaging of the appearance or disappearance of optical features,
primarily used with model metal NPs, was extended to visualize and quantify in situ
the formation or dissolution of a variety of other materials, such as gas nanobubbles
(79–83) or ionic crystals (84), and holds promise for high-throughput monitoring of
structural deformations of nanoelectrocatalysts under operating conditions (85). The
technique can be easily extended to the study of various phase formation processes as
long as they can be triggered electrochemically, either by direct electrodeposition or
indirectly by local electrolysis.
3.2.2 Electrochemical conversion
During the electrochemical or redox conversion of an NP, the change in the redox
state is often associated with a change in its optical properties, such as fluorescence,
absorption, or scattering cross-section. Different optical microscopies can distinguish
the initial and final states of redox conversion of individual NPs. Gradual changes in
composition can even be monitored in situ and in real time, revealing mechanistic
pathways at the single NP level (Ag examples in Figure 2a).
The conversion of Ag-based metallic NPs into Ag+ salt nanocrystals is evidenced
under dark-field microscopy by a decrease in the intensity of the light they scatter
without, however, reaching total signal extinction. A spectrometer inserted at the end
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of the optical path, or a hyperspectral camera, completed the pure optical imaging
with their UV-vis spectrum, providing information on the composition and conversion
mechanisms in solutions of Ag+ precipitating agents (77, 78, 86–88).
The transformation of metallic NPs, e.g., Ag, into more noble metal NPs, e.g., Au, by
galvanic replacement, a popular redox reaction in colloidal synthesis, was followed in
situ under Au3+ solution flow by dark-field microscopy. The optical transients are also
characterized by a sudden drop in the scattering signal but observed after a waiting
time of variable duration. The broad distribution of waiting times confirms the
gradual transformation of solutions. The difference between single vs. ensemble NP
behaviors suggests that the transformation is kinetically limited by the stochastic
formation of a void in the NP lattice (broad distribution). Once the void is formed, the
NP transformation is rapid (sudden drop) and diffusion-limited (89). Other
mechanistic indications were identified, such as the role of precipitating Cl- or the NP
ligand shell (90, 91).
The methodology is applicable to nanomaterials used for energy storage or
conversion. The refractive index of LiCoO2 NPs decreases linearly with the amount of
Li-expelled ions, allowing imaging of their electrochemical (de)lithiation by refractive
index-sensitive microscopies (92). From the optical intensity fluctuations of
individual NPs recorded during cyclic lithiation/delithiation voltammetry or
nanoimpact experiments (93), Wang and coauthors quantified the dynamics of Li-ion
diffusion with an optically inferred current sensitivity of 50 fA.
Similarly, for supercapacitor applications, the insertion/deinsertion of alkali ions into
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electrochromic NPs, such as WO3 or Prussian blue (PB), was imaged under
bright-field transmission (94–98). The evolution of the light transmitted by individual
NPs (Figure 2c) analyzed according to the Beer-Lambert law allows quantification of
their conversion rate. For some WO3 NPs, slower and less complete insertion
dynamics, even more pronounced for NP aggregates, were observed, which suggests
irreversible trapping of Li+ at the NP-NP or NP-electrode interfaces. The
heterogeneity of ionic nanocrystal-electrode contacts has also been highlighted when
potassium ions are inserted into electrochromic PB nanocubes (99). Sputtering an
ultrathin layer of Pt onto electrode materials, as is often done in SEM, reconnects and
renders all nanocubes electroactive and avoids erroneous conclusions in establishing
structure-activity relationships.
3.2.3 Electrocatalytic systems
3.2.3.1. Probing molecular intermediates
Quantification of the electrocatalytic activity of single NPs is achieved by probing the
molecular products or intermediates of the reaction by fluorescence microscopy or
surface-excited Raman spectroscopy (SERS), sometimes with single-molecule
sensitivity (see Section 2.4). These microscopies mainly use a redox molecular probe
(commonly phenoxazine dyes such as resorufin), in which one of the redox forms is
luminescent or Raman active, or use pH-sensitive probes.
By adapting the strategy developed for photocatalysis (100), fluorescence microscopy
allowed imaging and evaluating (i) the deactivation of Pt/C electrocatalysts during the
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hydrogen oxidation reaction (101) or (ii) the 2- vs. 4-electron reduction pathways of
O2 by magnetite NPs (102).
Willets et al. imaged by SERS the local activity and distribution of reaction potentials
on Ag NP aggregates for the 2-electron conversion of Nile blue (103, 104). A recent
work suggests possible extension to the direct detection of valuable reaction products
such as CO2 and its reduction products (105).
Electrochemiluminescence involves redox and electrochemical reactions that can be
activated by Au NPs (106), resulting in NP visualization. Under the oxidizing
conditions of electrochemiluminescence, the reaction is quickly deactivated (fading
image) owing to Au oxide formation, which was prevented using Janus Au-Pt NPs
(107).
3.2.3.2. Probing entities transformation
Owing to the sensitivity of the LSPR of a metallic NP to its free-electron density,
Mulvaney et al. (108) proposed monitoring the shift in LSPR wavelength by
spectroscopic scattering microscopies to image and quantify the flow of (few)
electrons during (dis)charging of Au NPs. The method has since been used to probe
any (electro)chemical reaction that would perturb the electron density of NPs (109,
110) to evaluate the rate of oxidation of ascorbic acid by O2 (111).
The LSPR is also influenced by the refractive index of the chemical environment of
the NPs, which allows imaging the electrocatalytic reactions that modify it, as
illustrated by Long et al. (112) during the oxidation of H2O2 on Au nanorods. The
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nanorod surface, first oxidized to Au hydroxide/oxide, is then reduced back to Au
while oxidizing H2O2, with the two activities represented by different LSPR shifts.
Local refractive index changes following electrocatalytic reactions at nonplasmonic
NPs can also be detected, but they must be deposited on an optically active electrode
that allows such sensitivity, e.g., in plasmonic-based or interference scattering
microscopy. The concept, developed by Tao et al. (28), to obtain local
electrochemical activities of heterogeneous electrodes allowed the establishment of
hydrogen evolution reaction (HER) CV at single Pt NPs (113).
Electrocatalysis of the HER or oxygen evolution reaction (OER), which is critically
important in energy applications, often leads to the formation of gas bubbles. Bubbles
are thought to nucleate and grow in regions supersaturated by gas molecules. Optical
microscopies that can probe bubble production at the micro (114) and nanoscale (115)
enable the identification of the most active catalysts.
Nanobubbles (NBs) were revealed during HER on Au nanoplates by TIR fluorescence
microscopy by tracking the adsorption of a single rhodamine molecule at the
electrolyte-gas interface (Figure 3a). The collected fluorescence intensity additionally
allows NB size estimation (79, 80). Due to their low refractive index, NBs are also
directly detected by label-free microscopy (Figure 3b). Optically undetectable Au-Pt
NPs were revealed from their electrogenerated NBs (116). Superresolution
plasmonic-based (117) or interference-reflection (82) microscopy allows rapid
dynamic mapping of NB nucleation sites on Au or ITO electrodes. The optical
response further provides a dynamic estimation of the size and shape of NBs (or
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contact angle), suggesting that Pt nanocatalysts were rapidly electrically isolated by
NBs (83), and their continuous growth proceeded through spill-over (79).
INSERT FIGURE 3
3.2.4. Superlocalizing physical changes
Controlling operando the deformation or structural alteration of NPs is crucial for the
longevity of electrocatalytic energy conversion devices (85). Such information, often
hidden in electrochemical analysis, except for some stochastic collision experiments
(56), is within the reach of optical imaging via spatial superlocalization in 2D or 3D
of the centroid of the optical pattern of NPs (118) during electrochemical solicitation.
The edge-tracking procedure suggested by Tao and coauthors allows localization of
the contours and thus evaluation of an apparent size of objects with a size comparable
or higher than the diffraction limit (119). The strategy highlighted the reversible
breathing of single Co(OH)2 particles while being electrochemically probed in the
OER region (120).
The motion and orientation of individual electroactive pseudo-2D graphene
microplatelets were optically tracked as they approached and collided with a
microelectrode (Figure 3c). The latter yields variations in the overall electroactive
surface area that correlate with transient variations in the electrochemical current
(121) until they rearrange themselves flat on the electrode (122). The dynamics of the
process are obtained from the rate of angular motion of the platelet.
25
3.3 PROBING SUBNANOENTITIES
Optical imaging can image beyond the single NP resolution and probe
transformations at the subunit level. First, an NP is separated into two classical
subunits: the shell, which is in contact with the outer environment, can exhibit a
different reactivity than the NP core. With the help of dedicated optical models, the
contribution of these subunits can be revealed optically. In a second approach,
anisotropic or localized electrochemical processes inside an NP are revealed by a
superlocalization approach (2.2.4).
3.3.1 Surface alteration
Imaging the shift in LSPR wavelength of plasmonic NPs by spectroscopic DFM
allows probing (electro)chemical conversion of the NP shell. The strategy developed
to monitor the deposition of Ag on strongly scattering Au nanostars (123) allows the
detection of the underpotential deposition of Ag on various shaped Au nanocrystals.
An LSPR shift of a few nm corresponds to submonolayer deposition. An optical
voltammogram (Figure 4a) is obtained from the LSPR frequency variations, revealing
the influence of the crystallographic facet orientation on the Ag electrodeposition
potential (124). The electrochemical conversion of the Ag shell to AgCl was
monitored in a similar manner (Figure 2a). The reaction intermediates distinguished
optically from their different plasmonic coupling modes suggest propagation of the
Ag/AgCl interface between the Au core and the chloride electrolyte interface (125).
The reversible electrochemical de/amalgamation of Au NPs by Hg (126, 127) was
26
similarly imaged. It first involves saturation of the NP surface by Hg atoms before
their slow diffusion into the core. The reverse process operates in the same way but
with a slower solid diffusion rate for the expelled Hg atoms.
Similarly, Link, Landes et al. (110) distinguished the reversible physical adsorption of
chloride ions on the surface of an Au NP from the irreversible formation of an Au
chloride shell. Then, they generalized the method to probe the electrochemical
adsorption dynamics of various molecules or anions (128).
As discussed in 2.2, the chemical reactivity of the NP surface, not restricted to
plasmonic NPs, was probed using refractive index-based techniques. Plasmonic-based
imaging has been used to differentiate between surface and bulk oxidation (or
reduction) for Au NPs or electrodes (129, 130). Interferometric scattering microscopy
has been more recently introduced to electrochemical studies, although it shows high
imaging sensitivity of various charge transfer processes. Although at the LiCoO2
microparticle but in a real Li-ion battery configuration, it allowed operando dynamic
imaging of local Li ion flow during (des)insertion (Figure 4b), revealing how its
heterogeneity can alter battery operation (131). It could also identify the restructuring
of electrochemical double layers at ITO or Cr nanostructures in iodide electrolyte
(132).
INSERT FIGURE 4
3.3.2 Centroid superlocalization
Edge-tracking procedures (119) were used to evaluate the electrochemical
27
deformation of gold nanowires (133) and graphene sheets (44) due to surface stress
and Pauli repulsion, respectively.
The superlocalization of AgCl NPs colliding with a cathodically biased electrode
revealed conversion in multiple motion-reaction steps attributed to loose electrical
connections (Figure 2a). Chloride ions are released locally in multiple steps, each
creating a limited silver metal inclusion within the NP and propelling the NP to a
nearby reactive site (86).
If the displacement of the NP PSF over distances greater than the NP dimension
reveals their physical motion, a slight spatial fluctuation can be attributed to an
asymmetric transformation of the NPs, highlighting the presence of inactive zones
within the NP.
A shift in the centroid of Ag NPs was observed by Willets et al. during their oxidation
(134), suggesting asymmetric dissolution limited by the asymmetric formation of a
nonconductive surface oxide (Figure 2a).
Similarly, the reduction of single PB NPs (98) to Prussian white is not always
complete. The position of the optical centroid depends on the intermediates formed
locally and therefore fluctuates during the conversion (Figure 4c). A microscopy
approach was then proposed to evaluate the propagation of the reaction along the
vertical direction. Since no vertical propagation is detected, conversion is thought to
occur via a shell-to-core model.
Ultimately, Wang and coauthors showed that Fourier transform analysis of the optical
28
images enables pushing the superlocalization procedure down to subnanometer
accuracy. By optically imaging the charge separation in Au nanorods subjected to
periodic capacitive charge-discharge cycles, they detected a periodic subnanometer
centroid shift (Figure 4d), suggesting heterogeneous charge accumulation on the Au
surface (96). This unprecedented resolution should unlock the label-free observation
of nanoscale local surface chemistry or the manipulation of single NP local reactive
sites (135).
3.4 SINGLE MOLECULE ELECTROCHEMISTRY
Molecular electrochemistry is a very broad subject involving many new concepts and
techniques, some with an unprecedented level of spatial resolution giving it a new
impetus, such as for the establishment of structure-function relationships at the
single-molecule scale (14, 15, 59, 136–138). In 1995, Fan and Bard first demonstrated
a single-molecule electrochemical measurement. This uses the concept of current
amplification by catalytic redox cycles, which involves repeating the oxidation and
reduction events of a molecule placed between two electrodes (139). To date, high
spatial resolution optical approaches have been developed to capture the intermediate
states of the electrochemical reaction of a single electroactive molecule, such as
surface-enhanced Raman spectroscopy and single-molecule fluorescence
spectroscopy (14, 16, 140, 141). Very recently, these methods have been transposed
to electrochemiluminescence imaging by Feng and coauthors (Figure 5a), showing
how, in complete darkness but by precise control of the chemical reaction between
29
electrogenerated reactants, electrodes can turn-on single-photon chemiluminescent
reactions (13). This opens a fascinating area of molecular electrochemistry and
electroanalytical research.
INSERT FIGURE 5
More abundant literature uses the former two optical approaches detailed here. The
tiny optical response during the transition of different redox states of a single
molecule can be followed to probe the electrochemical dynamics. This reveals the
intrinsic mechanism of electron transfer reactions in homogeneous solutions, enabling
the fundamental understanding of the electrochemistry of single molecules.
3.4.1 Surface-enhanced Raman spectroscopy
Single molecule surface-enhanced Raman spectroscopy (SM-SERS) can be used to
directly probe individual heterogeneous electrochemical events in a single molecule
(8, 14, 16, 138, 142). It provides fundamental information about structural changes
and specific behavior of a surface reactive site with respect to a redox couple or to
understand molecular electron transfer mechanisms and intracellular dynamics in
analytical chemistry. Plasmonic nanostructures locally enhancing the Raman intensity
are defined as "hot spots" in SERS in which vibrational information is captured to
determine the redox transient states of target electroactive molecules.
In 2010, SM-SERS was applied for the first time to discover electrochemical events in
a bianalyte system combining two dye molecules, rhodamine-6G (R6G) and Nile blue
30
(143). In an open-frame electrochemical cell, the presence of distinct imprinting
modes of a molecule at a hot spot was captured to address redox (on-off) events. In
addition, a thought-provoking question was answered as to whether the "average"
behavior of a bulk system can be recovered from the events of a single molecule.
Identical local conditions can only be extracted from measurements of a single
molecule without averaging electrical or optical properties. In parallel, Van Duyne
implemented SM-SERS to study single electron transfer events (O+ 1e! ⇄ R) of the
dye molecule R6G adsorbed on a silver NP under nonaqueous conditions (144). The
broad local distribution of reduction potentials can be attributed to variations in
molecular orientations and variations in the local surface site or chemical potential of
the R6G-Ag bonding units.
Recently, Wilson and Willets demonstrated the superresolution imaging strategy of
SM-SERS with sub-10 nm accuracy by establishing the spatial relationship between
the centroid of the SERS emission and the corresponding maximum intensity (104,
145, 146). Using this approach, they visualized the specific redox potentials at
different adsorption sites of individual Nile blue molecules on colloidal Ag NPs. The
reversible trajectories of the centroid of the molecules on the surface of the NPs
during a redox cycle were attributed to the location-dependent potentials of the single
electroactive molecule, where the SERS intensity modulates the activation and
deactivation states with oxidation and reduction processes.
31
3.4.2 Single molecule fluorescence spectroscopy
The synergistic coupling of electrochemistry with single molecule fluorescence
spectroscopy (SMFS), via confocal laser scanning, TIR, or superresolution
microscopes (15, 59, 137, 147, 148), allows the study of heterogeneous electron
transfer events by simultaneously capturing a quantitative electrochemical signal and
in situ fluorescence images. The key feature of this coupling is to obtain both
temporally and spatially resolved information by following the electron transfer
process. The redox states of the electrofluorochromic compounds at the
single-molecule limit can be determined from the blinking (on/off states) of the
fluorescence response. As the electrode potential varies, the residence time constant in
each of the states (on/off, then ox/red) reflects the rate of the redox transformation and
thus the dynamics of the electrochemical reaction.
In 2006, Bard and Barbara demonstrated for the first time the possibility of studying
single-molecule electron transfer processes by spectroelectrochemistry (149). They
studied hole-injection oxidation events of single molecules of
poly-9,9-dioctylfluorene-cobenzothiadiazole (F8BT), a redox conjugated organic
polymer used in solar cells and flat panel displays, immobilized on an ITO electrode.
As oxidation quenches fluorescence, the electron transfer dynamics are studied as a
function of potential and illumination. If both the excited and ground states of F8BT
can be oxidized, only the ground state oxidation shows a narrow distribution of
fluorescence turn-off potential, revealing its half-wave potential.
32
The technique has been extended to the study of more conventional fluorescent probes
in bioimaging. Gooding et al. were the first to report reversible fluorescence switching
of bovine serum albumin (BSA)-conjugated Alexa Fluor 647 redox probes by TIRF
(150). The potential-modulated fluorescence of BSA-Alexa Fluor 647 immobilized on
ITO at the single protein level was studied by measuring the variation in the number
and intensity of fluorescent spots. The observed pH dependence indicates the
involvement of two-electron one-proton transfer in the fluorescence switching
mechanism.
Orrit and coauthors (151) studied the fluorescent readout of redox-sensitive methylene
blue probes at the single-molecule level enhanced by individual gold nanorods
(Figure 5b). MB, a common redox indicator for tissue staining and biosensing,
undergoes a reversible fluorescence change to form colorless methylene blue by
two-electron one-proton transfer. Time traces of the fluorescence flashing of a single
electrogenerated MB molecule are recorded at different potentials. The residence
times in the on/off states are evaluated by a step detection algorithm. The distribution
of these residence times at each potential is used to evaluate the half-wave potential of
single molecule electrochemical switching from the Nernst equation.
4. PERSPECTIVES AND CONCLUSION
This review has shown how advanced optical microscopies are now able to image a
33
wide range of electrochemical phenomena with unprecedented temporal and spatial
resolutions (below the diffraction limit), up to the single object level with subentity,
subnanoparticle or single molecule sensitivity. By providing quantitative descriptors
complementary to electrochemical signals, they have unraveled old problems while
revealing new ones in the different fields explored by electrochemistry (sensors,
electroanalysis, corrosion, electrocrystalization, energy conversion and storage,
electrocatalysis, etc.).
First developed through model systems, they are now shifting to materials and
configurations focused on real-world applications, where they can be exploited to
precisely locate heterogeneous electrochemical processes, distinguish domains
(electrode regions, nanoobjects, etc.) of different structure/composition and therefore
distinguish competing chemical routes, or identify the origin of problems to fix. As
definitely the most intuitive platform to see operando, optical microscopies should
become a routine electroanalytical tool to evaluate the performance of electroactive
materials and rationalize their design or degradation. An even deeper degree of
understanding can be reached from their simple implementation with complementary
structural and chemical analyses such as spectroscopy (UV-vis or Raman, as well as
the promising surface-enhanced IR) or within multicorrelative microscopies
combining, e.g., local electrochemical probes and in situ TEM. Particularly,
approaches combining optical visualization within complementary electrochemical
local probing, e.g. by SECCM (57, 60), will enable the generation of large sets of
correlated optical and electrochemical data. It should become a powerful approach for
34
benchmarking wide range of electrochemical situations.
The generalization of these explorations to broader electrochemical situations also
implies seeing with greater sensitivity (e.g., iSCAT (10, 132) or photothermal
microscopes) more rapidly in more complex media (seeing through fog is within
reach) or in real-world systems (optical fiber explorations). It is also necessary to
generalize the nature of current collectors (optoelectrodes) providing sensitive optical
detection ensuring homogeneous (electro)chemical contact with the objective of
studying minimal electrocatalytic activity, for which transparent carbon- or
graphene-based electrodes are promising. Finally, the thousands of data per image,
even tenfold with complementary spectroscopic data, promise to unlock many
structure-function understandings. The use of artificial intelligence will be crucial to
achieve faster automated postprocessing, e.g., object identification by deep learning
(152), or recognition of electrochemical behavior and for the removal of unnecessary
information to optimize data storage and processing in real time.
ACKNOWLEDGMENTS
F.K. acknowledges support from the European Union’s Horizon 2020 Research and
Innovation Programme under Marie Skłodowska-Curie MSCA-ITN Single-Entity
Nanoelectrochemistry, SENTINEL [812398]. J.-F.L. and F.K. acknowledge the
Université de Paris and CNRS for financial support. W. Wang and H. Wang
acknowledge the National Natural Science Foundation of China (Grants 21925403,
21904062 and 21874070) and the Excellent Research Program of Nanjing University
35
(Grant ZYJH004) for financial support.
36
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FIGURE CAPTIONS
Figure 1. (a) Schematic illustration of the plasmonic-based electrochemical
impedance imaging technique of action potentials in single neurons. A micropipette is
patched on single neurons cultured on the surface to trigger action potentials, which
are recorded by patch clamp electronics and plasmonic imaging. Adapted with
permission from Reference (22). Copyright 2017, John Wiley & Sons. (b) Schematic
illustration of a typical electrochemiluminescence imaging technique for visualizing
the latent fingerprints on electrode surfaces with negative and positive modes.
Adapted with permission from Reference (29). Copyright 2012, John Wiley & Sons.
(c) Superlocalization of Zn dendrite nucleation and growth monitored by dark-field
microscopy in a Zn aqueous battery configuration. Adapted with permission from
Reference (54). Copyright 2021, Elsevier.
Figure 2. (a) Summary of optical microscopy studies reporting single silver-based NP
electrochemistry grouped into three main categories: growth, dissolution and
conversion, with corresponding references. Adapted with permission from Reference
(3). Copyright 2021, John Wiley & Sons. (b) Quantitative light scattering monitoring
of silver NP deposition and stripping voltammetry. The optical intensity transients
(extracted in ROIs) are quantitatively converted into single NP currents and
optovoltammograms. Adapted with permission from Reference (64). Copyright 2018,
John Wiley & Sons. (c) Optical transmittance monitoring of WO3 NP electrochemical
conversion. The different optical transients reveal heterogeneous Li-ion insertion in
46
single NPs and aggregates. Adapted with permission from Reference (94). Copyright
2019, American Chemical Society.
Figure 3. (a) Imaging of gas nanobubbles nucleating and growing upon
electrocatalytic water splitting in the vicinity of four nanocatalysts by TIR
fluorescence microscopy. Adapted with permission from Reference (79). Copyright
2018, National Academy of Sciences. (b) H2 nanobubbles equivalently detected at
single Pt nanocatalysts by interference reflection microscopy. The optical data are
further exploited to estimate the evolution of the contact angle of single NBs during
the growth process. Adapted with permission from Reference (83). Copyright 2021,
American Chemical Society. (c) Optical tracking of the graphene platelet impact
event and further dynamic rotation at a polarized microinterface. Adapted with
permission from Reference (122). Copyright 2021, American Chemical Society.
Figure 4. Operando optical screening enables subentity studies. (a) Optically inferred
voltammetry tracking facet-dependent underpotential deposition of silver atoms on
single gold truncated octahedral nanocrystals. Adapted with permission from
Reference (124). Copyright 2020, CC BY 4.0. (b) Optical image showing the front of
the phase transition in the LixCoO2 cathode particle, from which the charge-discharge
dynamics are revealed operando in a real Li-ion battery. Adapted with permission
from Reference (131). Copyright 2021, Springer Nature. (c) The optical centroid
47
motion of a single Prussian blue NP during oxidation/reduction cycles reveals local
transformations or inactive sites. Because the centroid is moving back to its initial
position, the conversion is reversible. Adapted with permission from Reference (98).
Copyright 2021, CC BY-NC 3.0. (d) Ultimate tracking resolution: the electrochemical
charging of single Au nanorods results in subnanometer optical centroid motion
owing to local electron accumulation. Reproduced with permission from Reference
(153). Copyright 2019, American Chemical Society.
Figure 5. Electrochemistry with a single-molecule fluorescence readout. (a) Principle
of single electrochemiluminescent event observation enabling single molecule
luminescence imaging, without illumination, of arrays of nanoband electrodes. The
dilution of both the dye and coreactant electrogenerated intermediate imposes a single
reaction event, i.e. the formation of the excited dye molecule further emitting a single
photon, located where the reaction was initiated during the image snapshot. Adapted
with permission from Reference (13). Copyright 2021, Springer Nature. (b)
Schematic showing the local fluorescence emission under plasmon-enhanced
photoactivation of the electrogenerated fluoroactive form of a single immobilized
electrofluorophore. The red/ox proportion of the single molecule is obtained from
blinking (right part) of the fluorescence signal at a fixed electrode potential. Adapted
with permission from Reference (151). Copyright 2021, John Wiley & Sons.
48
FIGURE 1
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FIGURE 2
50
FIGURE 3
51
FIGURE 4
52
FIGURE 5